CN117285637A

CN117285637A - Anti-idiotype antibody and application thereof

Info

Publication number: CN117285637A
Application number: CN202311563003.1A
Authority: CN
Inventors: 方鹏; 曹雨霞; 游猛; 施磊; 冯庆君; 朱晓红
Original assignee: Jiangsu T Mab Biopharma Co ltd; Jiangsu Maiweikang New Drug Research And Development Co ltd
Current assignee: Jiangsu T Mab Biopharma Co ltd; Jiangsu Maiweikang New Drug Research And Development Co ltd
Priority date: 2023-11-22
Filing date: 2023-11-22
Publication date: 2023-12-26
Anticipated expiration: 2043-11-22
Also published as: CN117285637B

Abstract

The application relates to the field of antibody medicines, in particular to an anti-idiotype antibody and application thereof. The present application provides an anti-idiotype antibody suitable for specifically binding to an anti-RANKL antibody or a derivative thereof, said anti-idiotype antibody comprising a heavy chain variable region comprising CDR-H1, CDR-H2 and CDR-H3 of the heavy chain variable region having the amino acid sequences SEQ ID nos. 1-3, respectively, and a light chain variable region comprising CDR-L1, CDR-L2 and CDR-L3 of the light chain variable region having the amino acid sequences SEQ ID nos. 4-6, respectively. The anti-idiotype antibody has high binding force and high specificity of the anti-RANKL antibody, and can be used for detecting the content of the anti-RANKL antibody medicine in a sample, thereby playing an important role in the pharmacokinetic and pharmacodynamic analysis of the anti-RANKL antibody medicine.

Description

Anti-idiotype antibody and application thereof

Technical Field

The application relates to the field of antibody medicines, in particular to an anti-idiotype antibody and application thereof.

Background

Receptor activators of NF kappa-B ligand (RANKL), also known as osteoprotegerin ligand (OPGL) and tumor necrosis factor ligand superfamily member 11 (TNFSF 11), are members of the TNF superfamily (Anderson DM, et al, nature 390 (6656): 175-179). RANKL can be expressed in soluble form on the cell surface (Findlay DM, et al, osteodoros Int 22 (10): 2597-2602). The formation, activation and survival of osteoclasts are mediated by binding to the receptor RANK on osteoclasts and their precursors (Hsu H, et al Proc Natl Acad Sci U S A1999 Mar 30;96 (7): 3540-2545). There is also increasing evidence that tumor cells interact within the bone, stimulating receptor activators of the RANK-RANKL system, leading to cancer-induced bone destruction (Roodman GD, N Engl J Med 2004; 350:1655-1664).

Early animal studies demonstrated the potential of RANKL as a therapeutic target for the treatment of osteoclast-mediated bone loss (Lacey DL, et al, cell 1998;93:165-176;Ann E.Kearns,et.al,Endocrine Reviews 29 (2): 155-192). Animal studies and clinical trials using OPG Fc fusion protein, an endogenous RNAKL inhibitor, provide strong evidence that RANKL is critical in bone remodeling (Ann e.kearns, et al Endocrine Reviews (2): 155-192;Bekker PJ,J Bone Miner Res.2001;16:348-360).

An anti-idiotype antibody is an antibody directed against a specific epitope group (idiotype) on the variable region of an antibody molecule, which is capable of specifically recognizing and binding to a unique site of another antibody. The anti-idiotype antibody is one of main tool reagents in the development process of antibody medicaments, and is mainly used for: 1) The anti-idiotype antibody can be used for detecting the content (free form, combined form and total amount) of various antibody drugs in blood because of different specific binding epitopes, thereby playing an important role in Pharmacokinetic (PK) and Pharmacodynamic (PD) analysis of the antibody drugs; 2) Anti-idiotype antibodies also play an important role in immunogenicity evaluation as positive controls for anti-drug antibody (ADA) detection.

Therefore, development of an anti-idiotype antibody with high binding force and high specificity against a RANKL antibody is required to be applied to aspects such as antibody drug development and immunogenicity evaluation.

Disclosure of Invention

In view of the above-mentioned drawbacks of the prior art, an object of the present application is to provide an anti-idiotype antibody and its application for solving the problems in the prior art.

To achieve the above and other related objects, a first aspect of the present application provides an anti-idiotype antibody adapted to specifically bind to an anti-RANKL antibody or a derivative thereof, the anti-idiotype antibody comprising a heavy chain variable region comprising CDR-H1, CDR-H2 and CDR-H3 having the amino acid sequences SEQ ID nos. 1-3, respectively, and a light chain variable region comprising CDR-L1, CDR-L2 and CDR-L3 having the amino acid sequences SEQ ID nos. 4-6, respectively.

The amino acid sequence is specifically as follows:

CDR-H1：DSSMK（SEQ ID No:1）

CDR-H2：DINQNNGDSFKNQKFKD（SEQ ID No:2）

CDR-H3：EQYGSIYVEYSWFAY（SEQ ID No:3）

CDR-L1：KAMQNVDTSVA（SEQ ID No:4）

CDR-L2：SASYQRS（SEQ ID No:5）

CDR-L3：QQYKVYPFT（SEQ ID No:6）。

"anti-idiotype antibody" refers to an antibody that specifically recognizes/binds to the idiotype of the antibody used to make them (i.e., has the idiotype of the variable region of the antibody used to make them as an epitope), which is capable of mimicking/rebuilding the epitope recognized by the antibody used to make them. The anti-idiotype antibody is an anti-idiotype antibody of the anti-RANKL monoclonal antibody, can recognize specific antigen epitope on a variable region of the anti-RANKL monoclonal antibody molecule, and has high binding force and high specificity to the RANKL monoclonal antibody. "affinity" or "affinity" refers to the strength of a non-covalent interaction between an immunoglobulin molecule (i.e., an antibody) or fragment thereof and an antigen. "specific" refers to determining the presence or absence of a protein in a heterogeneous population of proteins and/or other organisms, e.g., the binding reaction of an anti-idiotype antibody of the invention with an anti-RANKL antibody protein. Thus, under the specified conditions, a particular ligand/antigen binds to a particular receptor/antibody and does not bind in significant amounts to other proteins present in the sample.

In the anti-idiotype antibody provided by the application, the heavy chain variable region also comprises framework regions HFR1, HFR2, HFR3 and HFR4 with amino acid sequences of SEQ ID No. 7-10 respectively, and the specific sequences are as follows:

HFR1：EVQLQQSGPELVKPGASVKMSCKASGYTFT（SEQ ID No:7）

HFR2：WVRQSHGKSLEWIG（SEQ ID No:8）

HFR3：RATLTVDKSSSTAYMQLNSLTSEDSAVYYCSR（SEQ ID No:9）

HFR4：WGQGTLVTVSA（SEQ ID No:10）。

in the anti-idiotype antibody provided by the application, the light chain variable region also comprises framework regions LFR1, LFR2, LFR3 and LFR4 with amino acid sequences of SEQ ID No. 11-14 respectively, and the specific sequences are as follows:

LFR1：DIVMTQSQKFMSTSVGDRVSVTC（SEQ ID No:11）

LFR2：WYQQKPGQSPKTLIY（SEQ ID No:12）

LFR3：GVPDRFTGSGSGTDFTLTISNVQSEDLAEYFC（SEQ ID No:13）

LFR4：FGSGTKLEIK（SEQ ID No:14）。

in the anti-idiotype antibody provided by the application, the amino acid sequence of the heavy chain variable region of the anti-idiotype antibody is shown as SEQ ID No. 15; the amino acid sequence of the light chain variable region of the anti-idiotype antibody is shown in SEQ ID No. 16. The specific sequence is as follows:

the heavy chain variable region is as follows, wherein the CDR regions are indicated in bold:

EVQLQQSGPELVKPGASVKMSCKASGYTFTDSSMKWVRQSHGKSLEWIGDINQNNGDSFKNQKFKDRATLTVDKSSSTAYMQLNSLTSEDSAVYYCSREQYGSIYVEYSWFAYWGQGTLVTVSA（SEQ ID No:15）；

the light chain variable region is as follows, with CDR regions indicated in bold:

DIVMTQSQKFMSTSVGDRVSVTCKAMQNVDTSVAWYQQKPGQSPKTLIYSASYQRSGVPDRFTGSGSGTDFTLTISNVQSEDLAEYFCQQYKVYPFTFGSGTKLEIK（SEQ ID No:16）。

in the anti-idiotype antibody provided by the application, the amino acid sequence of the heavy chain comprises a sequence shown as SEQ ID No. 17, the variable region of the heavy chain is underlined, the CDR regions are bolded, and the specific sequence is as follows:

EVQLQQSGPELVKPGASVKMSCKASGYTFTDSSMKWVRQSHGKSLEWIGDINQNNGDSFKNQKFKDRA TLTVDKSSSTAYMQLNSLTSEDSAVYYCSREQYGSIYVEYSWFAYWGQGTLVTVSAAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK（SEQ ID No:17）。

in the anti-idiotype antibody provided by the application, the amino acid sequence of the light chain comprises a sequence shown as SEQ ID No. 18, the variable region of the light chain is underlined, the CDR regions are bolded, and the specific sequence is as follows:

DIVMTQSQKFMSTSVGDRVSVTCKAMQNVDTSVAWYQQKPGQSPKTLIYSASYQRSGVPDRFTGSGSG TDFTLTISNVQSEDLAEYFCQQYKVYPFTFGSGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC（SEQ ID No:18）。

among the anti-idiotype antibodies provided herein, the anti-idiotype antibodies are full-length antibodies, fab fragments, F (ab) ₂ Fragments, fv fragments, scFv fragments or single domain antibodies. In one embodiment of the present application, the anti-idiotype antibody is a full length antibody. Full length antibodies are glycoproteins comprising at least two heavy (H) chains and two light (L) chains, the heavy and light chains being composed of two chainsAnd (3) sulfur bond connection. Each heavy chain is composed of a heavy chain variable region (abbreviated as VH) and a heavy chain constant region. The heavy chain constant region is composed of three domains, namely CDR-H1, CDR-H2 and CDR-H3. Each light chain is composed of a light chain variable region (VL) and a light chain constant region. The light chain constant region is composed of one domain CL. VH and VL regions can also be divided into hypervariable regions called Complementarity Determining Regions (CDRs) which are separated by more conserved Framework Regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains comprise binding domains that interact with antigens. The constant region of an antibody may mediate the binding of an immunoglobulin to host tissues or factors, including various immune system cells (e.g., effector cells) and the first component of the traditional complement system (C1 q).

Among the anti-idiotype antibodies provided herein, the anti-idiotype antibody is a blocking antibody. Blocking antibodies refer to antibodies that bind to a protein of interest directly affecting the function of the protein, such as blocking the binding of an adhesion protein to a receptor ligand. The blocking antibodies of the present application recognize the anti-RANKL antibodies, do not recognize the control human IgG, and block the binding of the anti-RANKL antibodies to RANKL, and are therefore considered blocking antibodies.

The anti-idiotype antibody provided by the application is an antigen blocking antibody, can be specifically combined with an anti-RANKL antibody and a derivative thereof, and can block the combination of the anti-RANKL antibody and the derivative thereof with the RANKL. anti-RANKL antibodies and derivatives thereof can specifically bind RANKL, thereby preventing interaction of RANKL with RANK. The anti-RANKL antibody may be, for example, desquamation. Deshumab is a fully human monoclonal antibody, and can be used as an RANKL inhibitor for human treatment. The anti-RANKL antibody derivative may be, for example, an antibody-conjugated drug (ADC). The ADC combines the high specificity of the monoclonal antibody medicine with the high activity of the small molecular cytotoxic medicine, so as to improve the targeting property of the tumor medicine and reduce the toxic and side effects.

A second aspect of the present application provides a method for detecting an anti-RANKL monoclonal antibody drug or derivative thereof in a sample, comprising:

(a) Contacting a sample with the anti-idiotype antibody described above under conditions sufficient to form an immune complex between the anti-idiotype antibody described above and an anti-RANKL monoclonal antibody drug or derivative thereof;

(b) Detecting the presence of said immune complex, wherein the presence of said immune complex indicates the presence of an anti-RANKL monoclonal antibody drug or derivative thereof in said sample.

The method for detecting the anti-RANKL monoclonal antibody drug or the derivative thereof in the sample can be a disease diagnosis or treatment method, and also can be a non-disease diagnosis or treatment method, and particularly can be used for example in the aspects of anti-RANKL antibody drug resistance detection, anti-RANKL antibody drug immunogenicity evaluation, drug screening, drug evaluation and the like.

In the method provided herein, in step (a), the substance formed by the binding of the antigen and the corresponding antibody is referred to as an immune complex.

In step (a), an anti-idiotype antibody or an anti-RANKL monoclonal antibody drug or a derivative thereof is immobilized directly or indirectly to a solid support. In some embodiments, the solid support is selected from the group consisting of acetate membranes, test tubes, EP tubes, multiwell plates, microplate wells, and microspheres.

In step (b), the detection is performed by mixing a known anti-idiotype antibody with the sample, and after a period of time, if an immune complex formation occurs, it is indicated that the corresponding anti-RANKL monoclonal antibody drug or its derivative is present in the sample. If no expected phenomenon occurs, it is indicated that no corresponding anti-RANKL monoclonal antibody drug or derivative thereof is present in the sample. Based on the detection of the sample, a clinical prodrug generation analysis can be performed against RANKL monoclonal antibody drugs or derivatives thereof, such as absorption, distribution and transport of the anti-RANKL monoclonal antibody drugs or derivatives thereof in vivo. When the quantitative detection is carried out on the sample, the concentration of the anti-idiotype antibody added in the reaction is in a functional relation with the concentration of the immune complex, and the content of the anti-RANKL monoclonal antibody drug or the derivative thereof in the sample can be estimated according to the generation amount of the immune complex: under certain reaction conditions, the concentration of the anti-idiotype antibody added is certain, and the amount of immune complex generated by the reaction is in direct proportion to the amount of the corresponding anti-RANKL monoclonal antibody drug or the derivative thereof contained in the sample to be detected. The amount of the anti-RANKL monoclonal antibody drug or derivative thereof may be determined by detecting the amount of the label. The content of the anti-RANKL monoclonal antibody drug or the derivative thereof in the sample can be calculated by using an experimental standard curve. Such as immune one-way diffusion test, immune turbidimetry test and enzyme-linked immunosorbent assay. Thus, pharmacokinetic analysis of anti-RANKL monoclonal antibody drugs or derivatives thereof, e.g. the law of the in vivo drug concentration over time, may be performed.

In the method provided by the present application, in step (b), detecting the presence of an immune complex is accomplished by the following method:

i. via a detectable label linked directly or indirectly to an anti-idiotype antibody;

via other reagents that bind to anti-idiotype antibodies or anti-RANKL monoclonal antibody drugs or derivatives thereof, directly or indirectly linked to a detectable label;

realization via another of the aforementioned anti-idiotype antibodies, the other anti-idiotype antibody being directly or indirectly linked to a detectable label and recognizing the other idiotype;

this is achieved via an additionally added anti-RANKL monoclonal antibody drug or derivative thereof, to which a detectable label is directly or indirectly attached.

In some embodiments, i.is that when an anti-idiotype antibody binds to an anti-RANKL monoclonal antibody drug or derivative thereof to form an immune complex, the presence of the immune complex can be recognized by detection of the detectable label due to the detectable label carried directly or indirectly by the anti-idiotype antibody.

In some embodiments, the other agent in ii. May be, for example, a secondary antibody that is homologous to the anti-idiotype antibody, is different from the anti-RANKL monoclonal antibody drug or derivative thereof, and thus may bind to the anti-idiotype antibody and not to the anti-RANKL monoclonal antibody drug or derivative thereof. The secondary antibody can be used as a detection antibody for detecting the combination of an anti-idiotype antibody and an anti-RANKL monoclonal antibody drug or a derivative thereof, if the combination generates an immune complex, a detectable label carried by the secondary antibody can be identified, and if the anti-idiotype antibody is not combined with the anti-RANKL monoclonal antibody drug or the derivative thereof, the secondary antibody can not detect the existence of the immune complex.

In some embodiments, iii.and iv.can be, for example, a double antibody sandwich method.

The principle of the medium double anti-sandwich method is that an anti-idiotype antibody is combined on a solid phase carrier to form a solid phase antibody, then the solid phase antibody is combined with a corresponding anti-RANKL monoclonal antibody drug or a derivative thereof in a sample to form an immune complex, the immune complex is washed, the anti-idiotype antibody is additionally added, a detectable label is directly or indirectly connected with the additionally added anti-idiotype antibody, the anti-idiotype antibody drug or a derivative thereof is combined with the immune complex to form an anti-idiotype antibody-anti-RANKL monoclonal antibody drug or a derivative thereof, the substrate is added for color development, and the situation of the anti-RANKL monoclonal antibody drug or the derivative thereof in the sample is judged.

The principle of the medium double antibody sandwich method is that an anti-RANKL monoclonal antibody drug or a derivative thereof is combined on a solid phase carrier to form a solid phase antibody, then the solid phase antibody is combined with an anti-idiotype antibody to form an immune complex, the anti-RANKL monoclonal antibody drug or the derivative thereof is additionally added after washing, a detectable marker is directly or indirectly connected with the additionally added anti-RANKL monoclonal antibody drug or the derivative thereof, the anti-RANKL monoclonal antibody drug or the derivative thereof is combined with the immune complex to form the anti-RANKL monoclonal antibody drug or the derivative thereof-anti-idiotype antibody-anti-RANKL monoclonal antibody drug or the derivative complex, the color of the base is developed, and the condition of the anti-idiotype antibody is judged.

In some embodiments, the detectable label comprises: fluorescent markers, chromogenic markers, reporter genes, localization signals, and the like. In particular, detectable labels include enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron emitting metals, and non-radioactive paramagnetic metal ions. More than one tag may also be included. The labels used to label antibodies depend on the particular detection method used, e.g., ELISA (enzyme-linked immunosorbent assay) and immunofluorescence techniques, immunoradiometric techniques, immunoenzymatic techniques and immunocolloidal gold techniques, immunohistochemical staining (tissue) samples, flow cytometry, and the like. Suitable labels are well known to those skilled in the art for detection methods known in the art.

In a third aspect the present application provides an isolated nucleotide molecule encoding an anti-idiotype antibody as described above. The full length nucleotide sequence of the anti-idiotype antibody or fragment thereof of the present application can be obtained generally by PCR amplification, recombinant or synthetic methods. One possible approach is to synthesize the sequences of interest by synthetic means, in particular with short fragment lengths. In general, fragments of very long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them. In addition, the coding sequences for the light and heavy chains may be fused together to form an anti-idiotype antibody.

In a fourth aspect the present application provides a vector comprising an isolated nucleotide molecule as hereinbefore described. "vector" refers to a polynucleotide that can be transcribed and translated into a polypeptide when introduced into a suitable host cell. Vectors of the present invention generally refer to a variety of commercially available expression vectors well known in the art, and may be, for example, bacterial plasmids, phage, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors.

In a fifth aspect, the present application provides a host cell comprising the vector or genome as defined above, incorporating the isolated nucleotide molecule as defined above. Any cell suitable for expression of an expression vector may be used as a host cell, for example, the host cell may be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells.

In a sixth aspect, the present application provides a kit comprising an anti-idiotype antibody as described above, an isolated nucleotide molecule as described above, a vector as described above, or a host cell as described above.

In the kit provided by the application, the kit is an anti-RANKL antibody detection kit or an anti-RANKL antibody immunogenicity detection kit. The anti-idiotype antibody contained in the kit can be combined with the anti-RANKL antibody, so that the anti-RANKL antibody detection kit is used for directly detecting the anti-RANKL antibody, and the anti-RANKL antibody immunogenicity detection kit is used as a positive control for detecting the anti-RANKL antibody.

The kit of the application can be a colloidal gold immunoassay kit, a chemiluminescent kit, a radioimmunoassay kit, an enzyme-linked immunoassay kit or a fluorescent immunoassay kit. The kit may comprise an anti-idiotype antibody, an isolated nucleotide molecule, a vector, a host cell, or a composition contained therein for direct use. Meanwhile, the kit can also comprise reagents such as conventional buffer solution, washing solution, coating solution and the like which are necessary for detection.

The seventh aspect of the present application provides the use of the aforementioned anti-idiotype antibody, the aforementioned isolated nucleotide molecule, the aforementioned vector, the aforementioned host cell, or the aforementioned kit for the preparation of an anti-RANKL antibody detection product, a positive control for an anti-RANKL antibody resistance detection product, a positive control for an anti-RANKL antibody drug immunogenicity detection product, or a positive control for an anti-RANKL antibody neutralizing antibody.

In the application provided by the application, the anti-RANKL antibody detection product is used for qualitative or quantitative analysis of the anti-RANKL antibody or derivatives thereof. Further, the anti-RANKL antibody detection products were: the product of the pharmacokinetics analysis or pharmacodynamics analysis of the antibody.

An anti-RANKL antibody derivative may refer to an anti-RANKL antibody modified with a functional group having an antigen-specific recognition ability and binding ability or higher reactivity similar to those of an unmodified antibody. Functional groups for modification include, but are not limited to, fluorescent dyes, drugs, contrast agents, bridging molecules, polyethylene glycols, carbohydrates, lipids, nucleic acids, proteins, peptides, peptide analogs, and metal chelators. The anti-RANKL antibody derivative can also be a bispecific or multispecific antibody formed by coupling with other antibodies.

Pharmacokinetics (pharmacokinetics) is abbreviated as pharmacokinetics and is mainly studied as a dynamic change of the treatment of drugs (dispersion) by the body. Including absorption, distribution, biochemical transformation (or metabolism) and excretion of drugs in the body, especially the law of the change of blood concentration with time. Metabolism of drugs is related to age, sex, individual difference, genetic factors of humans, and the like. Pharmacodynamics and pharmacokinetics share common influencing factors such as dosage, pharmaceutical benefits and adverse reactions. Pharmacodynamics in particular emphasizes the dose-response relationship, i.e. the relationship between drug concentration and the effect of its corresponding organism (whether negative or positive). The anti-idiotype antibody can be used for detecting the change rule and the metabolic condition of the anti-RANKL antibody in a pharmacokinetics analysis test and a pharmacodynamics analysis of the anti-RANKL antibody.

One possible mechanism for the development of resistance to anti-RANKL antibodies is that the body contains antibodies against RANKL antibodies, thereby preventing the binding of anti-RANKL antibodies to RANKL or accelerating the in vivo clearance rate of anti-RANKL antibodies, and finally affecting the efficacy. The anti-RANKL antibody drug resistance detection product is an antibody used for detecting the anti-RANKL antibody, and the anti-idiotype antibody can be combined with the anti-RANKL antibody with high specificity and can block the combination of the anti-RANKL antibody and the RANKL antibody drug resistance detection product, so the anti-RANKL antibody drug resistance detection product can be used as a positive control of the anti-RANKL antibody drug resistance detection product.

An anti-RANKL antibody neutralizing antibody refers to an antibody that binds to an anti-RANKL antibody and blocks its antigen binding function. The anti-idiotype antibody can be combined with the anti-RANKL antibody and can block the combination of the anti-RANKL antibody and the RANKL, so the anti-idiotype antibody can be used as a positive control of the detection of the neutralizing antibody of the anti-RANKL antibody.

The anti-idiotype antibodies, isolated nucleotide molecules, vectors, host cells, or kits of the present application can specifically bind to RANKL antibodies and thus can be used as a positive control for binding agents to detect RANKL antibodies.

Compared with the prior art, the beneficial effects of this application are:

1. the anti-idiotype antibody has high binding force and high specificity of the anti-RANKL antibody, and can be used for detecting the content of the anti-RANKL antibody medicine in a sample, thereby playing an important role in the pharmacokinetic and pharmacodynamic analysis of the anti-RANKL antibody medicine.

2. The anti-idiotype antibody has good performance test, can be used as positive control for immunogenicity ADA analysis, and plays an important role in immunogenicity evaluation.

3. The anti-idiotype antibody is used as a target blocking type anti-idiotype antibody, and the target blocking type antibody can be used as a neutralizing antibody analysis positive control of an anti-RANKL monoclonal antibody.

Drawings

FIG. 1 is a standard graph of quantitative detection of anti-RANKL humanized monoclonal antibodies.

Fig. 2 is a graph of anti-idiotype antibody ADA analysis.

FIG. 3 is a graph of anti-idiotype antibody neutralizing antibody analysis (ligand binding analysis).

FIG. 4 is a graph of anti-idiotype antibody neutralizing antibody analysis (cell analysis).

Detailed Description

In order to make the objects, technical solutions and advantageous effects of the present application clearer, the present application is further described below with reference to examples. It should be understood that the examples are presented by way of illustration only and are not intended to limit the scope of the application. The test methods used in the following examples are conventional, unless otherwise indicated, and other advantages and effects of the present application will be readily apparent to those skilled in the art from the disclosure herein.

The inventors of the present application have found an anti-idiotype antibody and its use through extensive research and study, and completed the present application on the basis of this.

The present application is further illustrated by the following examples, which are not intended to limit the scope of the present application.

Example 1

Preparation of anti-idiotype antibodies

1. Animal immunization and antibody production

Pepsin (Sigma, cat# P6887) enzyme-cleaved anti-RANKL humanized monoclonal antibody (namely Deshu monoclonal antibody, manufactured by Miwei (Shanghai) Biotech Co., ltd.) and the Fc region of the antibody was removed to prepare F (ab) ₂ And (3) a sample. F (ab) to be obtained ₂ The sample was used as an immunogen for immunized animals, with 5 Balb/c cellsMice were immunized 3 times each with 3 weeks of each cycle. The serum of the immunized mice is required to be specifically combined with the corresponding immunogen, and the titer of the antiserum is more than 1:10,000.

Spleen cells of 1-2 mice with highest titers among immunized mice are selected for fusion with SP2/0 myeloma cells. Amplifying and culturing hybridoma cells, taking supernatant, detecting titer by ELISA, and detecting antibody subtype; cells positive for anti-RANKL humanized monoclonal antibodies and negative for irrelevant human IgG were selected for cloning. To ensure the positive rate of hybridoma cell strain and the stable production of antibody, 3-4 clones were carried out, each clone was separated by 1-2 weeks, and after 3-4 times of 100% positive clones, the cells were inoculated into mice after expanded culture to prepare ascites, thus obtaining the anti-idiotype antibody. The specificity of hybridoma supernatant was detected by indirect ELISA method during cloning, i.e. the murine monoclonal antibody in supernatant was only bound to the anti-RANKL humanized monoclonal antibody, but not to the unrelated human IgG, and finally 1 strain of the unique monoclonal antibody (clone number S-56-7) was obtained, the results are shown in Table 1.

TABLE 1 hybridoma supernatant specificity characterization results

2. Target competitive and noncompetitive testing

The following conditions were satisfied by detecting 1 strain of antibody produced by hybridoma cells:

1) Target non-blocking antibodies: detecting by ELISA method, recognizing anti-RANKL antibody, not recognizing control human IgG, and not blocking the binding of anti-RANKL antibody and RANKL (ELISA plate coated antigen, adding ELISA plate after incubation of anti-RANKL antibody and prepared anti-idiotype antibody, detecting by anti-human IgG secondary antibody, and signal value is not reduced compared with the incubation group without anti-idiotype antibody (PBS group);

2) Target blocking antibodies: the ELISA method is used for detection, the anti-RANKL antibody is identified, the control human IgG is not identified, the binding between the anti-RANKL antibody and the RANKL can be blocked (ELISA plate coated antigen, the anti-RANKL antibody is added into the ELISA plate after being incubated with the prepared anti-idiotype antibody, the anti-human IgG secondary antibody is used for detection, and the signal value is obviously reduced compared with that of an incubation group (PBS) without the anti-idiotype antibody).

The results are shown in Table 2, and the target blocking property of the anti-idiotype antibody was judged by comparing the difference of OD values of the added antibody group and the non-added antibody group. S-56-7 is a target blocking antibody.

TABLE 2 results of target competitive and noncompetitive testing

Example 2

Sequencing of anti-idiotype antibodies

1. Experimental procedure

And (3) amplifying and culturing the hybridoma cells, extracting total RNA of the cells, and carrying out reverse transcription to obtain cDNA. And (3) PCR amplification to obtain heavy chain and light chain variable region genes of the antibody, sequencing and performing bioinformatic analysis of the sequence, and eliminating nonfunctional antibody genes. Antibodies are named according to their hybridoma cell clone of origin, and the amino acid sequences of the heavy and light chains are shown below. The variable regions of the heavy and light chains are shown underlined and the CDR regions (complementarity determining regions) of the heavy and light chains, defined according to the KABAT naming system, are shown bolded.

2. Sequencing results

S-56-7 complete antibody sequences

Heavy chain sequence:

EVQLQQSGPELVKPGASVKMSCKASGYTFTDSSMKWVRQSHGKSLEWIGDINQNNGDSFKNQKFKDRA TLTVDKSSSTAYMQLNSLTSEDSAVYYCSREQYGSIYVEYSWFAYWGQGTLVTVSAAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK（SEQ ID No:17）

light chain sequence:

DIVMTQSQKFMSTSVGDRVSVTCKAMQNVDTSVAWYQQKPGQSPKTLIYSASYQRSGVPDRFTGSGSG TDFTLTISNVQSEDLAEYFCQQYKVYPFTFGSGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC*（SEQ ID No:18）

the antibody can be obtained by conventional genetic engineering to obtain an isolated monoclonal antibody, for example, by synthesizing the gene of the antibody, constructing a recombinant vector, transfecting 293 cells, carrying out recombinant expression, and purifying.

Example 3

Application of Anti-idiotype antibody of Anti-RANKL antibody in non-clinical and clinical drug analysis

1. Use of anti-idiotype antibodies in non-clinical drug generations

In the embodiment, an enzyme-linked immunosorbent assay (ELISA) method is used, and the application of the anti-idiotype antibody in the invention in non-clinical drug generation analysis is examined by taking cynomolgus monkey serum drug generation analysis as an example, taking target protein recombinant human RANKL extracellular protein as a capture reagent and taking an anti-idiotype antibody as a detection reagent.

Test method

Coating and sealing: diluting recombinant human RANKL extracellular protein (ACRO, product number RAL-H5240) to 2 mug/mL with coating liquid, and standing overnight at 2-8 ℃; the ELISA plate is patted dry, and the ELISA plate is washed 3 times by a washing buffer solution; the ELISA plate is dried, 3% BSA/PBST is added, and the ELISA plate is incubated for 2 hours at room temperature; the blocked ELISA plates were then dried by pipetting, and the ELISA plates were rinsed 3 times with wash buffer.

Standard curve and quality control dilution: anti-RANKL humanized mab (manufactured by Jiangsu meweikang new drug development limited) was diluted with 100% pooled monkey serum (pooled monkey serum, PMS) to prepare standard curves with the following concentrations: 10000ng/ml (ULOQ, upper limit of quantitation), 5000ng/ml, 2500ng/ml, 1250ng/ml, 625ng/ml, 312.5ng/ml, 156.25ng/ml (LLOQ, lower limit of quantitation), 78.12ng/ml (anchor point). The blank (0 ng/ml) was used as part of the standard curve, but was not involved in the standard curve fitting. The 5 quality control concentrations were also prepared with 100% pooled monkey serum: 10000ng/mL (ULOQ, upper limit of quantification), 8000ng/mL (HQC, high concentration quality control), 2000ng/mL (MQC, medium concentration quality control), 400ng/mL (LQC, low concentration quality control), 156.25ng/mL (LLOQ, lower limit of quantification).

Diluting and adding a sample to be tested: after 50 times of pre-dilution of the monkey serum sample to be tested, the standard curve sample and the quality control product with 1% BSA/PBST respectively, adding the samples into an ELISA plate according to 100 μl/well, providing a multiplex well, standing at room temperature and incubating for 2h.

Adding a detection antibody: after incubation, the ELISA plates were patted dry and washed 6 times with wash buffer; antigen blocking idiotype antibody (S-56-7) was diluted to 2. Mu.g/ml with 1% BSA/PBST, and then added to ELISA plates, and incubated at room temperature for 1h.

Adding a detection secondary antibody: after incubation, the ELISA plates were patted dry and washed 6 times with wash buffer; goat anti-mouse IgG HRP-labeled secondary antibody (Jackson ImmunoResearch, cat. No. 115-035-003) was diluted 20000 times with 1% BSA/PBST and added to ELISA plate, and incubated at room temperature for 1h.

Color development: after incubation, the ELISA plates were patted dry and washed 6 times with wash buffer; adding TMB substrate (KPL, cat. No. 5120-0047) equilibrated to room temperature into ELISA plate, and incubating at room temperature in dark place for about 10 min; 1M H is added ₃ PO ₄ The reaction was terminated.

Reading a plate: after the end of the color reaction, the OD value at the wavelength of 450nm was read with 650nm as a reference wavelength.

Concentration calculation: and performing four-parameter fitting on the standard curve concentration and the OD value, and calculating the drug concentration of the sample to be detected by using a fitting equation.

The method is used for detecting the concentration of the anti-RANKL humanized monoclonal antibody in monkey serum, the standard curve is shown in figure 1, and the antigen blocking antibody (S-56-7) can be used as a detection antibody for drug generation analysis and has higher detection sensitivity and signal response value. The accuracy and precision of the method are examined by using the antibody, the result is shown in table 3, and the antibody can accurately quantify the drug concentration in the serum of the cynomolgus monkey.

TABLE 3 accuracy and precision (S-56-7)

2. Application of anti-idiotype antibody in detection of drug immunogenicity ADA

The immune response of a therapeutic protein drug may affect the pharmacokinetics, pharmacodynamics, safety and effectiveness of the drug. The adverse reactions of most drugs are caused by humoral immune mechanisms-mediated immune responses, and detection of drug-resistant antibodies (ADA) is a major method for assessing drug immunogenicity. This example mainly examined the use of anti-idiotype antibodies in the detection of immunogenic ADA.

Test procedure

Coating and sealing: diluting the anti-RANKL humanized monoclonal antibody (manufactured by Jiangsu Meikang New drug development Co., ltd.) to 1 mug/ml with a coating solution, and standing at 2-8 ℃ overnight; the ELISA plate is patted dry, and the ELISA plate is washed 3 times by a washing buffer solution; the ELISA plate is dried, 3% BSA/PBST is added, and the ELISA plate is incubated for 2 hours at room temperature; the blocked ELISA plates were then dried by pipetting, and the ELISA plates were rinsed 3 times with wash buffer.

Preparing a sample to be tested: anti-idiotype antibody (S-56-7) was diluted to 10. Mu.g/ml with 10% pooled monkey serum (10% PMS) as starting concentration, 3-fold gradient dilution for 7 spots and 10% PMS as 0 spot.

Sample adding: anti-RANKL humanized monoclonal antibody was labeled according to the instructions with EZ-LINK NHS-LC-LC-Biotin (Thermo, cat # 21343) to give anti-RANKL antibody-Biotin, which was diluted to 50ng/ml with 1% BSA/PBST. The diluted anti-idiotype antibody sample and anti-RANKL antibody-Biotin were added to ELISA plates at 50. Mu.l/well, and the wells were multiplexed and incubated at room temperature for 2h.

Adding SA-HRP: after incubation, the ELISA plates were dried by pipetting, and washed 6 times with wash buffer. SA-HRP (southern Biotech, cat. No. 7105-05) was diluted 1:5000 with 1% BSA/PBST and added to ELISA plates and incubated for 1h at room temperature.

Color development: after incubation, the ELISA plates were dried by pipetting, and washed 6 times with wash buffer. TMB substrate (KPL, cat. No. 5120-0047) equilibrated beforehand to room temperature was added to ELISA plates and incubated at room temperature for about 10min in the absence of light. 1M H is added ₃ PO ₄ The reaction was terminated.

Test results

As shown in FIG. 2, the anti-idiotype antibody S-56-7 has good performance test, and the signal value and the antibody concentration show good dose-response relationship, and can be used as positive control of ADA analysis.

3. Application of anti-idiotype antibody in detection of drug neutralizing antibody

Neutralizing activity refers to the ability of an anti-drug antibody to inhibit the biological activity of a drug. Neutralizing antibodies can block the binding of the product to its target or interfere with receptor/ligand binding, interfering with the in vivo activity of the drug. This example evaluates the neutralizing activity capacity of anti-idiotype antibodies from non-cell based ligand binding assays and cell based neutralization activity assays, respectively.

The method comprises the following steps: ligand binding assays

Coating and sealing: diluting recombinant human RANKL extracellular protein (ACRO, product number RAL-H5240) to 1 mug/mL by using coating liquid, and standing overnight at 2-8 ℃; the ELISA plate is patted dry, and the ELISA plate is washed 3 times by a washing buffer solution; the ELISA plate is dried, 3% BSA/PBST is added, and the ELISA plate is incubated for 2 hours at room temperature; the blocked ELISA plates were then dried by pipetting, and the ELISA plates were rinsed 3 times with wash buffer.

Preparing a sample to be tested: anti-idiotype antibody (S-56-7) was diluted to 10. Mu.g/ml with 10% pooled monkey serum (10% PMS) as starting concentration, 2-fold gradient dilution for 11 spots and 10% PMS as 0 spot.

Sample adding: anti-RANKL humanized monoclonal antibody (manufactured by Jiangsu Meikang New drug development Co., ltd.) was labeled with EZ-LINK NHS-LC-LC-Biotin (Thermo, cat # 21343) according to the specification to give anti-RANKL antibody-Biotin, which was diluted to 50ng/ml with 1% BSA/PBST. The diluted anti-idiotype antibody sample and anti-RANKL antibody-Biotin were added to ELISA plates at 50. Mu.l/well, and the wells were multiplexed and incubated at room temperature for 2h.

Color development: after incubation, ELISA plates were patted dry and washedThe ELISA plates were washed 6 times with buffer. TMB substrate (KPL, cat. No. 5120-0047) equilibrated beforehand to room temperature was added to ELISA plates and incubated at room temperature for about 10min in the absence of light. 1M H is added ₃ PO ₄ The reaction was terminated.

Test results

As shown in FIG. 3 and Table 4, S-56-7 is a target blocking type anti-idiotype antibody, which can block the binding of anti-RANKL humanized monoclonal antibody and RANKL protein in a concentration-dependent manner. Thus, the target blocking antibody S-56-7 can be used as a positive control in an anti-RANKL humanized monoclonal antibody neutralizing antibody assay.

TABLE 4 anti-idiotype antibody neutralizing antibody analysis data (ligand binding)

The second method is as follows: cell analysis

Cell preparation:

taking 1 bottle of Cell RANK-GloResponse NF- κB-RE-luc2P HEK293 Cell Line (MW 03 RGA Cell, constructed by Jiangsu Meikang New drug development Co., ltd.) transformed with RANK gene, NF- κB-RE and luciferase reporter gene, collecting cells, and adjusting Cell density to 10×10 ⁴ Mu.l/well of each of the cells was inoculated into 96-well white cell plates at 37℃with 5% CO ₂ The cell culture incubator was incubated overnight. The HEK293 cell surface over-expresses a receptor RANK protein of RANKL, when the RANKL is combined with the RANK on the cell surface, an NF- κB signal path in the cell is activated, the expression of a luciferase reporter gene (luc 2P) is further increased, and the expression level of luciferase can be realized by detecting a luminescence signal after adding a luciferase substrate. When the anti-RANKL humanized monoclonal antibody is added, the binding of the RNAKL and the RANK is blocked, and the final signal is inhibited; when the S-56-7 antibody is added, the binding between the anti-RANKL humanized monoclonal antibody and the RANKL can be blocked, the binding between the RNAKL and the RANK can be recovered, and the final signal can be recovered.

Sample dilution and incubation

anti-RANKL humanized monoclonal antibody (manufactured by Jiangsu Meikang New drug development Co., ltd.) and recombinant human RANKL extracellular protein (company ACRO, cat. RAL-H5240) were diluted to 50ng/ml and 300ng/ml with DMEM medium containing 10% FBS, respectively. Anti-idiotype antibodies were diluted to 5000ng/ml with DMEM medium containing 10% fbs, followed by a further 3-fold dilution of 8 concentration gradients. Taking a clean centrifuge tube, adding diluted anti-idiotype antibody and anti-RANKL humanized monoclonal antibody into each tube in equal volume, and uniformly mixing.

Sample addition

Adding 50 μl/well of the anti-idiotype antibody and anti-RANKL humanized monoclonal antibody mixed solution into 96-well white cell plates paved with cells the day before adding 50 μl/well, adding diluted recombinant human RANKL extracellular proteins into each well plate, placing into a 5% CO2 cell incubator at 37 ℃, and reacting for 4-6 hours.

Color development and reading plate

Taking out the 96-well white cell plate, pouring out the culture medium in the plate, adding a working solution of Bright-Glo ™ Luciferase Assay System (Promega, product number E2620) into 50 mu l/well, and reacting for 5 minutes at room temperature in a dark place; in the luminence mode, the intersystem selection 500 (instrument default) reads RLU.

Test results

As shown in Table 5 and FIG. 4, the anti-idiotype antibody (S-56-7) was able to block the binding of the anti-RANKL humanized antibody to RANKL, and as the concentration of the anti-idiotype antibody increases, the anti-RANKL humanized antibody capable of binding to RANKL was lower, so that the more RANKL bound to RANK on the cells was, the NF- κB signaling pathway was activated after binding to RANK to initiate the expression of the reporter gene, and fluorescence was generated. Therefore, the strain of anti-idiotype antibody is a target blocking type and can be used as a positive control for neutralizing antibody analysis.

TABLE 5 anti-idiotype antibody neutralizing antibody analysis data (cell analysis)

In conclusion, the anti-idiotype monoclonal antibody capable of specifically recognizing the anti-RANKL humanized monoclonal antibody has the advantages of good uniformity and strong binding force, can be widely applied to the field of drug research and development, and is mainly applied to immunogenicity analysis, pharmacokinetic analysis and the like of antibody drugs.

The above embodiments are merely illustrative of the principles of the present invention and its effectiveness, and are not intended to limit the application. Modifications and variations may be made to the above-described embodiments by those of ordinary skill in the art without departing from the spirit and scope of the present application. Accordingly, it is intended that all equivalent modifications and variations which can be accomplished by persons skilled in the art without departing from the spirit and technical spirit of the present disclosure shall be covered by the claims of this application.

Claims

1. An anti-idiotype antibody adapted to specifically bind to an anti-RANKL antibody or a derivative thereof, said anti-idiotype antibody comprising a heavy chain variable region comprising CDR-H1, CDR-H2 and CDR-H3 having the amino acid sequences SEQ ID nos. 1-3, respectively, and a light chain variable region comprising CDR-L1, CDR-L2 and CDR-L3 having the amino acid sequences SEQ ID nos. 4-6, respectively.

2. The anti-idiotype antibody of claim 1, wherein the heavy chain variable region further comprises framework regions HFR1, HFR2, HFR3, and HFR4 having the amino acid sequences of SEQ ID Nos 7-10, respectively;

and/or the light chain variable region further comprises framework regions LFR1, LFR2, LFR3 and LFR4 of which the amino acid sequences are SEQ ID Nos. 11 to 14, respectively.

3. The anti-idiotype antibody of claim 1, wherein the amino acid sequence of the heavy chain variable region of said anti-idiotype antibody is shown in SEQ ID No. 15 and the amino acid sequence of the light chain variable region is shown in SEQ ID No. 16.

4. The anti-idiotype antibody of claim 1, wherein the heavy chain of the anti-idiotype antibody has an amino acid sequence as set forth in SEQ ID No. 17; the amino acid sequence of the light chain of the anti-idiotype antibody is shown as SEQ ID No. 18.

5. The anti-idiotype antibody of claim 1, wherein said anti-idiotype antibody is a full length antibody, fab fragment, F (ab) ₂ Fragments, fv fragments, scFv fragments or single domain antibodies.

6. The anti-idiotype antibody of claim 1, wherein the anti-idiotype antibody is a blocking antibody.

7. The anti-idiotype antibody of claim 1, wherein the anti-RANKL antibody and its derivatives are desquamation antibody and its derivatives.

8. A method for detecting an anti-RANKL monoclonal antibody drug or derivative thereof in a sample, the method comprising:

(a) Contacting a sample with the anti-idiotype antibody of any of claims 1-7 under conditions sufficient for the anti-idiotype antibody of any of claims 1-7 to form an immune complex with an anti-RANKL monoclonal antibody drug or derivative thereof;

(b) Detecting the presence of said immune complex, said presence of said immune complex indicating the presence of said anti-RANKL monoclonal antibody drug or derivative thereof in said sample.

9. The method according to claim 8, wherein in step (a), the anti-idiotype antibody or anti-RANKL monoclonal antibody drug or derivative thereof is immobilized directly or indirectly to a solid support.

10. The method of claim 9, wherein in step (b), detecting the presence of the immune complex is accomplished by:

i. via a detectable label directly or indirectly attached to the anti-idiotype antibody;

via a further agent binding to said anti-idiotype antibody or to said anti-RANKL monoclonal antibody drug or derivative thereof, said further agent being directly or indirectly linked to a detectable label;

realization via another anti-idiotype antibody according to any of claims 1 to 7, which is directly or indirectly linked to a detectable label and recognizes an additional idiotype;

via the additionally added anti-RANKL monoclonal antibody drug or derivative thereof, to which a detectable label is directly or indirectly attached.

11. An isolated nucleotide molecule encoding the anti-idiotype antibody of any one of claims 1-7.

12. A vector comprising the isolated nucleotide molecule of claim 11.

13. A host cell comprising the vector or genome of claim 12 having integrated therein the isolated nucleotide molecule of claim 11.

14. A kit comprising an anti-idiotype antibody according to any of claims 1 to 7, an isolated nucleotide molecule according to claim 11, a vector according to claim 12, or a host cell according to claim 13.

15. Use of an anti-idiotype antibody according to any of claims 1-7, an isolated nucleotide molecule according to claim 11, a vector according to claim 12, a host cell according to claim 13, or a kit according to claim 14 for the preparation of an anti-RANKL antibody detection product, a positive control for an anti-RANKL antibody resistance detection product, a positive control for an anti-RANKL antibody drug immunogenicity detection product, or a positive control for an anti-RANKL antibody neutralizing antibody.

16. The use according to claim 15, wherein the anti-RANKL antibody detection product is used for qualitative or quantitative analysis of anti-RANKL antibodies or derivatives thereof.

17. The use according to claim 16, wherein the anti-RANKL antibody detection product is: the product of the pharmacokinetics analysis or pharmacodynamics analysis of the antibody.